To date, pluripotent ES cells have not been described in the rat. It is possible, however, reproducibly to establish continuous diploid cell lines from rat pre-implantation embryos. The differentiation potential of these cells appears to be restricted to extraembryonic tissues. The primary objective of the proposed research is to develop a system for using these cell lines to transmit genetic modifications into the rat germline. To this end three routes will be addressed: (i) further modification of the derivation and culture protocol, notably by inhibition of specific intracellular signalling pathways, may enable derivation of true pluripotent stem cells directly from rat blastocysts; (ii) genetic manipulation, in particular, forced expression of the master transcription factor Oct-3/4, may be sufficient to maintain pluripotency in culture and/or convert established cells to a state of pluripotency; (iii) the diploid cells may be employed as donors for NT. Each of these studies will be facilitated by the use of transgenic rats and derivative cell lines that express the b-galactosidase reporter under control of the Oct-3/4 promoter. These transgenic rats will also be used to evaluate the possibility of establishing embryonic germ (EG) cells from the rat. In parallel with establishing protocols for cell line derivation and germline transmission, targeted genetic modification will be examined at two loci, Hprt and the rat orthologue of mouse Rosa26. The efficiencies of homologous recombination will be determined for isogenic replacement type vectors. These approaches should enable generation of rats that ubiquitously express green fluorescent protein (GFP) and/or beta-galactosidase reporters from one or other of these loci and thereby demonstrate the feasibility of precision genome engineering in the rat.